1. Field of the Invention
The present invention relates to a process for obtaining hirudin derivatives from E. coli secretor mutants, and to a hirudin derivative with the N-terminal amino-acid sequence (SEQ ID NO: 1).
2. The Prior Art
Hirudin is a polypeptide with 65 amino acids and was originally isolated from the leach Hirudo medicinalis. It acts as a highly specific inhibitor of thrombin by forming stable complexes with thrombin and, therefore, has many possible therapeutic uses, especially for anticoagulation therapy (F. Markquardt, Biomed. Biochim. Acta 44 (1985), 1007-1013).
The publication of the complete amino-acid sequence of hirudin (J. Dodt et al., FEBS LETTERS 165 (2), (1984), 180-184) was the prerequisite for the preparation of hirudin by recombinant DNA techniques and expression in microorganisms.
European Patent Application No. 158,564 (Transgene) discloses cloning vectors for the expression of hirudin or hirudin analogues in a host cell, especially a bacterial cell. The gene coding for hirudin is, in this case, obtained by cDNA synthesis starting from mRNA from the leach Hirudo medicinalis. Described, in particular, is a hirudin derivative with the N-terminal sequence (SEQ ID NO: 2) and processes for obtaining it.
European Patent Application No. 168,342 (Ciba Geigy) discloses DNA sequences which code for the natural amino-acid sequence of hirudin, wherein the N-terminal amino-acid sequence is (SEQ ID NO: 3). The expression of hirudin takes place intracellularly in the microorganisms E. coli and Saccharomyces cerevisiae. 
European Patent Application No. 171,024 (Hoechst AG) discloses a process for the genetic engineering for preparation of polypeptides with hirudin activity, in particular, in E. coli cells, wherein the cells are disrupted and the polypeptide with hirudin activity is obtained from the cell extract. A fusion protein portion which is present where appropriate can be deleted by proteolytic or chemical cleavage, and the liberated hirudin molecule can be purified.
German Patent Application No. 3,445,571 (GEN-BIO-TEC) relates to a DNA sequence which codes for a protein with the biological activity of hirudin, and to a process for obtaining such proteins from E. coli cells which are transformed with a suitable recombinant vector by lysis of the cells.
The paper by Bergmann et al (Biol. Chem. Hoppe Seyler 367 (1986), 731-740) also describes hirudin synthesis in E. coli. The hirudin is released from the cells by toluene treatment, with only low yields of about 500 ng/l A578 units of cells being achieved.
European Patent Application No. 200,655 (Transgene), European Patent Application No. 252,854 (Transgene), and European Patent Application No. 225,633 (Ciba Geigy) disclose the obtaining by secretion of proteins with hirudin activity from a eukaryotic host organism, especially yeast, wherein the expression takes place on a vector which contains a DNA sequence which contains a signal peptide upstream of the structural gene. The secretion of hirudin derivatives with the N-terminal sequence (SEQ ID NO: 3) and with the N-terminal sequence (SEQ ID NO: 2) in yeast is disclosed. In this case, yields of up to 100 mg/l are reported.
German Patent Application No. 3,900,626 (Hoechst AG) discloses a hirudin derivative with the N-terminal sequence (SEQ ID NO: 4). The expression takes place preferably in yeast, using the promoter and signal sequence of the yeast pheromone gene MFxcex1 for the expression and secretion of the hirudin derivative.
All the processes described above for preparing hirudin derivatives have disadvantages, however. Thus, when yeast is used as the host organism, and the hirudin is secreted into the culture medium, relatively high yields are obtained, but the cultivation of yeast cells takes longer and is more demanding than that of bacteria, for example, E. coli. However, on the other hand, in E. coli cells, the yield is relatively low, and/or complicated isolation processes are necessary on disruption of the cells.
Accordingly, it is an object of the present invention to develop a straightforward process for obtaining hirudin derivatives in which hirudin derivatives can be obtained in high yield from bacterial cells without entailing the necessity of disruption of the cells.
The present invention relates to a process for obtaining hirudin derivatives from E. coli secretor mutants which entails:
(1) construction of a recombinant vector on which there Is located the gene coding for a hirudin derivative downstream of a DNA section which codes for a bacterial signal peptide;
(2) transforming an E. coli secretor mutant with the recombinant vector constructed in step (1);
(3) cultivating the transformed cells in a medium; and
(4) obtaining the hirudin derivative from the medium.
The term xe2x80x9chirudin derivative,xe2x80x9d according to the present invention, refers to proteins which are derived from hirudin which act as thrombin inhibitors and have a specific activity of at least 10,000 AT-U/mg (antithrombin units) (Dodt et al., Biol. Chem. Hoppe Seyler 366 (1985), 379-385). The term xe2x80x9chirudin derivativexe2x80x9d also comprises fusion proteins with an N-terminal fusion portion which is up to about 50 amino acids long and can be partially or completely deleted by proteolytic or chemical cleavage, resulting in, as a cleavage product, a hirudin derivative of a specific activity of at least 10,000 AT-U/mg.
Preferably obtained by the process according to the invention are hirudin derivatives with the following N-terminal amino-acid sequence:
(X)mxe2x80x94Zxe2x80x94xe2x80x83xe2x80x83(SEQ ID NO: 5)
in which
Where m is greater than 0, the sequence X preferably contains a proteolytic or chemical cleavage site, particularly preferably at its end. If, for example, the last amino acid in the sequence X is an Arg residue, the fusion sequence X can be cleaved off by digestion-with trypsin (cleavage after Arg), and the active hirudin derivative can be purified. However, it is equally possible to cleave off the fusion portion using other known proteolytic enzymes or chemical cleavage reagents. If, for example, the amino-acid sequence of X terminates with a Met residue, the fusion protein can be cleaved by cleavage with cyanogen halides (E. Gross and B. Wittkop, J. Am. Chem. Soc. 82 (1961) 1510-1517). If, for example, the C-terminal amino-acid sequence of X contains the amino-acid sequence (SEQ ID NO: 6), the cleavage can be carried out with factor Xa (European Patent Application No. 25,190 and European Patent Application No. 161,973).
When m=0, in the process according to the invention, Z preferably represents Ala, Gln, His, Phe, Tyr, Gly, Ser, Asp or Asn, particularly preferably Ala, Gly, Ser, Asp or Asn. Maximum preference is given to a hirudin derivative in which m denotes 0 and Z represents Ala.
Thus, the present invention also relates to hirudin derivatives with the N-terminal sequence Axe2x80x94(SEQ ID NO: 5) in which A represents Ala, Gln, His, Phe, Tyr, Gly, Ser, Asp or Asn, preferably Ala, Gly, Ser, Asp or Asn. Maximum preference is given to a derivative with the N-terminal sequence (SEQ ID NO: 1). Surprisingly, it has been possible to obtain from this hirudin derivative in the culture supernatant of an E. coli secretor mutant up to above 2 g/l medium of active hirudin.
Another advantage of the process according to the invention is that, owing to the secretion of the hirudin derivative into the cell medium, the disulfide linkages of hirudin are correctly formed under the oxidative conditions of the medium.
According to the present invention, the term E. coli secretor mutants is intended to refer to E. coli strains which show massive protein secretion into the culture medium. A process for preparing these secretor mutants is disclosed in European Patent No. 338,410. The obtaining of suitable E. coli secretor mutants can start from, in particular, E. coli DS410 (DSM 4513) or E. coli BW7261 (DSM 5231). The particular E. coli strain is initially transformed with a plasmid which contains a DNA sequence coding for a secretable protein. The transformed E. coli strain is then subjected to a mutagenesis, for example, by treatment with N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine. This is followed by selection for suitable secretor mutant strains. If the secretable protein used is, for example, xcex1-cyclodextrin glycosyltransferase, secretor mutants can be recognized by resistance to the substance D-cycloserine, which is active on the cell wall. In addition, the secretion of xcex1-cyclodextrin glycosyltransferase (CGTase) brings about hydrolysis of the starch in the surrounding medium, which provides an additional option for selection of secretor mutants when an amylopectin azure medium is used.
Suitable as recombinant vectors for the present invention are vectors which either are able to integrate into the E. coli genome (for example, bacteriophagexcex) or are present extrachromosomally in the transformed E. coli cell (for example, plasmids). Plasmids are preferably used.
The gene construct which is on the recombinant vector and which codes for a protein consisting of signal peptide and the hirudin derivative is preferably under the control of an inducible promoter, particularly preferably of a trp-lac fusion promoter which is inducible by addition of lactose or IPTG (isopropylxcex2-D-thiogalactoside). In addition, a selection marker gene and, where appropriate, a lac repressor gene, should be present on the vector.
Suitable as a bacterial signal sequence which makes secretion of the hirudin derivative possible are, in principle, all known signal peptides which allow a permeation of the membrane of E. coli cells. Thus, also preferably used are signal peptides from Gram-negative bacteria as (for example, signal peptides of the following proteins of E. coli: outer membrane protein OmpA (DiRienzo et al, Ann. Rev. Biochem. 47 (1978) 481-532); alkaline phosphatase PhoA (Inouye et al, J. Bacteriol. 149 (1982) 434-439); LamB protein (Hedgpeth et al, Proc. Nat. Acad. Sci. USA 77 (1980) 2621-2625); Maltose binding protein MalE (Bedouelle et al, Nature 285 (1980) 78-81). The xcex1-CGTase signal peptide is particularly preferably used.
An example of a vector suitable for the process according to the invention is the plasmid pCM705 (FIG. 1), which can be obtained from the plasmid pCM703 disclosed in European Patent Application No. 383,410, by deletion of an NruI fragment which is about 1 kb long. This vector contains an ampicillin-resistance gene, the gene for the lac repressor and the CGTase gene with a section coding for the signal peptide at the 5xe2x80x2 end. A gene coding for a hirudin derivate is integrated into the vector pCM705 in such a way that there is intracellular production of a precursor molecule with the signal peptide of the xcex1-CGTase at its N-terminal end. The gene construct is under the control of the tac promoter. An E. coli secretor mutant strain can be transformed with the plasmid obtained in this way.
Positively transformed clones are cultivated in a shaken flask or in a fermenter. Induction by IPTG (isopropyl-xcex2-D-thiogalactoside) or lactose is carried out when an optical density (OD600) of about 1 is reached.
The progress of the production of the hirudin derivative is then determined by means of a thrombin inactivation test (Griesbach et al, Thrombosis Research 37, (1985), 347-350). The accumulation of fusion proteins is analyzed by HPLC chromatography (reversed phase). The proportion of fusion proteins can then be cleaved off, and the resultant active hirudin derivative can be purified.